A family of membrane embedded ATP-powered ion pumps, known as Secretory Pathway Ca2+, Mn2+-ATPases (SPCA), is conserved from yeast to human. SPCA pumps supply the Golgi lumen with ions essential for protein processing, sorting and glycosylation reactions. There is emerging evidence that SPCA pumps are also critically important for cytoplasmic Ca2+ signaling events and Mn2+ homeostasis. In this proposal, we will investigate three unique, physiologically distinct and clinically relevant functions of SPCA pumps. In Aim 1, we will test the hypothesis that SPCA1 contributes to manganese clearance through bile. We will use liver-specific, shRNA mediated knockdown of SPCA1 in a murine model to evaluate a role in manganese detoxification. Using a polarized, hepatocyte derived cell line, we will investigate the role of a novel Golgi Mn2+ sensor in SPCA1 trafficking, and identify molecular determinants for endosomal localization of SPCA1. In Aim 2, we will determine the mechanism of an unconventional interaction between SPCA2 and the Orai1 ion channel using fluorescence and electrophysiological approaches, as well as an innovative yeast expression strategy. N- and C-terminal SPCA2 domains with dominant negative or constitutively active properties will be evaluated for functional interactions with ion channels. In Aim 3, we seek to understand the physiological role of pump-channel interactions in eliciting robust calcium influx at the plasma membrane. We will follow up on preliminary studies showing that a calcium handling module of pumps, channels, buffers and sensors, including SPCA2, is coordinately induced upon lactation. A unique, 3-dimensional model of lactating mammary epithelial cells will be used to determine if SPCA2 interacts with and activates Orai channels for effective calcium secretion into milk. We will assess the function of a novel, truncated C-terminal SPCA2 transcript, specifically regulated by MIST1, a bHLH transcription factor that shows overlapping expression with SPCA2. Finally, we will examine how dysregulation of this calcium module in tumor cells contributes to distinct modes of cell proliferation and migration underlying cancer progression.